Although the genetic mechanisms of resistance to fluconazole have been studied in pathogenic yeasts belonging to the genus Candida, no studies have been conducted in C. neoformans. In the previous year, we studied fluconazole heteroresistant C. neoformans strains isolated from two AIDS patients and one HIV-negative patient. The two AIDS patients, one from Italy and one from the U.S. had been exposed to fluconazole while suffering from cryptococcosis. The HIV-negative cryptococcosis patient was in Israel who had never been treated with any antimycotic agent. Resistance to fluconazole in these strains lacked cut-off points and the majority of subpopulations obtained from a single colony was susceptible to the azoles while a fraction of the population was highly resistant. Purification of a susceptible population was not possible although purification of highly resistant populations were readily obtained by culturing the cells on media with azoles. The resistance in this highly homogeneous population was unstable and returned to original heteroresistance when the drug was removed from the culture media. This year we have studied their resistance pattern towards other triazoles and the effect of environmental conditions to the expression of resistance. The strains showed cross resistance to voriconazole but not all were cross resistant to itraconazole. These observations indicate that there is more than one mechanism leading to heteroresistance to azoles in C. neoformans. The pattern of heteroresistance was not affected by the pH or osmolarity of the medium but was influenced by temperature. The resistance appeared to be suppressed at 350C and was completely abolished at 400C. A genomic library was created from a subclone of the Italian isolate which is highly resistant to fluconazole (serotype D) and transformed into a sensitive wild type strain. In spite of repeated transformation attempt, no resistant transformants were obtained. This indicates that there may be multiple genes involved in the expression of heteroresistance in C. neoformans. A genomic library was created with the DNA extracted from resistant subpopulations and was used to transform a wild type strain homogeneously susceptible to azoles. No resistant transformants were obtained despite repeated attempts. These results also suggested that heteroresistance to azoles is regulated by more than one gene.